Development of a novel universal primer set with high sensitivity and specificity for detection of diatom.

The diatom test has long been used as an ancillary examination in the diagnosis of drowning. The conventional diatom test requires the handling of strong acids and depends on the investigator's skill and expertise. Several issues are also associated with the diatom test, such as sample contamination and diatom loss during testing. We have developed a novel primer set for detecting diatoms using real-time PCR. The primers were designed based on the rbcL gene as a marker, and the amplicon length was 68 bp. A high detection sensitivity (0.1 pg DNA) was achieved for all diatom species tested, and no amplification was observed using DNA from cyanobacteria or humans, demonstrating that the primer set has high specificity. Evaluation of the method's detection limit based on diatom counts using laser capture microdissection showed that the limit varied by species, ranging from 1 to 40 individuals. Diatoms were detected in various environmental water samples, including river and seawater, and in lung tissues from drowning autopsies. Although there are limitations to genus-level identification, this primer set was applicable to metagenomic analysis. Since this primer set is highly sensitive and specific and can detect diatoms even when their DNA is degraded, it has the potential for application in forensic practice. Furthermore, this method may enable validation of the premise underlying the diatom test-namely, that diatoms can disseminate to closed organs via the systemic circulation.